Morphologic and immunopathologic findings in myasthenia gravis and in congenital myasthenic syndromes.

Overwhelming evidence now supports Simpson's concept, originally proposed in 1960, that acquired myasthenia gravis (MG) is an autoimmune disease in which antibodies are directed against the nicotine postsynaptic acetylcholine receptor (AChR).1 An autoimmune pathogenesis of acquired MG implies that those myasthenic syndromes which occur in a congenital and familial setting may have a different, non-autoimmune basis. This paper focuses on ultrastructural, immunoelectron microscopic and cytochemical aspects of acquired autoimmune MG and some recently recognised congenital myasthenic syndromes.     

Myasthenia, thymoma, presynaptic antibodies, and a continuum of neuromuscular hyperexcitability.

BACKGROUND: Autoantibodies specific for the nicotinic acetylcholine receptor (AChR) of skeletal muscle impair neuromuscular transmission in myasthenia gravis (MG). Autoantibodies specific for alpha3 neuronal AChRs or voltage-gated potassium channels have been reported in patients with Isaacs syndrome, an acquired disorder of continuous muscle fiber activity characterized by neuromyotonia. OBJECTIVE: To report the neuromuscular autoantibody profiles of three patients with a syndrome of MG and neuromuscular hyperexcitability. RESULTS: All three patients reported here had clinical and electrophysiologic evidence of MG and neuromuscular hyperexcitability. None had neuromyotonia. Thymoma was proven in two patients and suspected in the third. One had MG and thymoma and subsequently developed cramp-fasciculation syndrome; MG and rippling muscle syndrome appeared simultaneously in the other two. All patients had muscle and neuronal AChR binding antibodies and striational antibodies. Only one had antibodies reactive with alpha-dendrotoxin-complexed potassium channels. CONCLUSIONS: The coexistence of cramp-fasciculation syndrome and acquired rippling muscle syndrome with MG, thymoma, and neuronal AChR autoantibodies suggests that there is a continuum of autoimmune neuromuscular hyperexcitability disorders related pathogenically to Isaacs syndrome. Manifestations of neuromuscular hyperexcitability may be altered and less apparent in the context of MG because of the coexisting defect of neuromuscular transmission.     

Monozygous twins with neuromuscular transmission defects at opposite sides of the motor endplate

Disorders affecting the postsynaptic side of the neuromuscular junction include autoimmune myasthenia gravis (MG) as well as some of the congenital myasthenic syndromes (CMS). Lambert-Eaton myasthenic syndrome (LEMS) is an acquired autoimmune neuromuscular disorder in which autoantibodies are directed against the presynaptic calcium channels. Here we describe two monozygous twin brothers: case 1 was diagnosed with an indeterminate form of acquired postsynaptic neuromuscular junction defect at age 32 and case 2 with LEMS at age 47. Case 1 presented clinically with mild generalized myasthenic weakness, neurophysiological examination revealed disturbed neuromuscular transmission along with probable myositis and serum analysis regarding antibodies against the acetylcholine receptor and muscle-specific tyrosine kinase was negative. Case 2 presented with proximal muscle fatigue accompanied by areflexia at rest and antibodies against the P/Q-type voltage-gated calcium channels were present. Neurophysiologically, case 2 had reduced baseline compound motor action potential amplitudes on neurography, decrement on low-frequency repetitive nerve stimulation (RNS) and pathological increment on high frequency RNS. To our knowledge this is the first case report of its kind and adds an intriguing contrast to the more common diagnosis of CMS in monozygous twins.     

Clinical evaluation and management of myasthenia gravis

Myasthenia gravis (MG) is a syndrome of fluctuating skeletal muscle weakness that worsens with use and improves with rest. Eye, facial, oropharyngeal, axial, and limb muscles may be involved in varying combinations and degrees of severity. Its etiology is heterogeneous, divided initially between those rare congenital myasthenic syndromes, which are genetic, and the bulk of MG, which is acquired and autoimmune. The autoimmune conditions are divided in turn between those that possess measurable serum acetylcholine receptor (AChR) antibodies and a smaller group that does not. The latter group includes those MG patients who have serum antibodies to muscle-specific tyrosine kinase (MuSK). Therapeutic considerations differ for early-onset MG, late-onset MG, and MG associated with the presence of a thymoma. Most MG patients can be treated effectively, but there is still a need for more specific immunological approaches.     

Acetylcholine receptors and myasthenia

Much progress has been made in the 26 years since initial studies of the first purified acetylcholine receptors (AChRs) led to the discovery that an antibody-mediated autoimmune response to AChRs causes the muscular weakness and fatigability characteristic of myasthenia gravis (MG) and its animal model, experimental autoimmune myasthenia gravis (EAMG). Now, the structure of muscle AChRs is much better known. Monoclonal antibodies to muscle AChRs, developed as model autoantibodies for studies of EAMG, were used for initial purifications of neuronal AChRs, and now many homologous subunits of neuronal nicotinic AChRs have been cloned. There is a basic understanding of the pathological mechanisms by which autoantibodies to AChRs impair neuromuscular transmission. Immunodiagnostic assays for MG are used routinely. Nonspecific approaches to immunosuppressive therapy have been refined. However, fundamental mysteries remain regarding what initiates and sustains the autoimmune response to muscle AChRs and how to specifically suppress this autoimmune response using a practical therapy. Many rare congenital myasthenic syndromes have been elegantly shown to result from mutations in muscle AChRs. These studies have provided insights into AChR structure and function as well as into the pathological mechanisms of these diseases. Evidence has been found for autoimmune responses even to some central nervous system neurotransmitter receptors, but only one neuronal AChR has so far been implicated in an autoimmune disease. Thus far, only two neuronal AChR mutations have been found to be associated with a rare form of epilepsy, but many more neuronal AChR mutations will probably be found to be associated with disease in the years ahead.     

Autoantibodies to low-density lipoprotein receptor-related protein 4 in myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction, where acetylcholine receptor (AChR), muscle-specific kinase (MuSK), and low-density lipoprotein (LDL) receptor-related protein 4 (Lrp4) are essential. About 80% and 0% to 10% of patients with generalized MG have autoantibodies to AChR and MuSK, respectively, but pathogenic factors are elusive in others. Here we show that a proportion of AChR antibody-negative patients have autoantibodies to Lrp4. These antibodies inhibit binding of Lrp4 to its ligand and predominantly belong to the immunoglobulin G1 (IgG1) subclass, a complement activator. These findings together indicate the involvement of Lrp4 antibodies in the pathogenesis of AChR antibody-negative MG.     

Pathophysiology of myasthenia gravis

Myasthenia gravis (MG) is arguably the best understood autoimmune disease, and its study has also led to fundamental appreciation of mechanisms of neuromuscular transmission. MG is caused by antibodies against the acetylcholine receptor (AChR), which produce a compromise in the end-plate potential, reducing the safety factor for effective synaptic transmission. It is clear that AChR antibody destruction of the postsynaptic surface is dependent on complement activation. A muscle-specific kinase has been recently found to be an antigenic target in MG patients without antibodies against the AChR. Autoantibody production in MG is a T-cell-dependent process, but how a breakdown in tolerance occurs is not known. In MG there is an interesting differential involvement of muscle groups, in particular, the extraocular muscles. This article reviews normal neuromuscular transmission, mechanisms of the autoimmune process of MG, and differential susceptibility of eye muscles to MG.     

Editorial introduction: from the structure and functions of the neuromuscular junction to the diseases

The neuromuscular junction has been recognized as a site for autoimmune and genetic disorders. Myasthenia gravis (MG) is mainly caused by postsynaptic nicotinic acetylcholine receptor (AChR) IgG1 antibodies that are directed against α-subunit 67-76 and 125-147 and activate complement. Thymic abnormalities are present in the autoimmune background. A proportion of MG patients without conformation-dependent AChR antibodies assayed by the cell-based method have muscle-specific tyrosine kinase (MuSK) antibodies which are largely IgG4 and partially IgG1. MuSK is activated by Dok-7 and Lrp4 (agrin receptor) and contributes to AChR clustering at the postsynaptic membrane via various kinase cascades in collaboration with Wnt-MuSK/Frizzled-Dishevelled signaling. Rapsyn interacts with MuSK-linked chaperones to stabilize postsynaptic architecture and also contributes to AChR phosphorylation. MG-associated thymomas express antigens that trigger antibody responses which play a part in disease generation and modification. Among these, ryanodine receptor-1 (RyR1; acts on sarcoplasmic Ca2+ release) antibodies cause muscle contractile weakness. Transient receptor potential canonical-3 (TRPC3) antibodies are also detected in thymoma-associated MG patients; they may participate in muscle contractile weakness because TRPC3 acts on RyR1, and may also impair the refill of sarcoplasmic Ca2+ stores since TRPCs contribute to the receptor-operated Ca2+ influx via the phospholipase C (PLC)-diacylglycerol (DAG) pathway in cooperation with the store-operated, STIM1/Orai1-mediated Ca2+ influx and TRPCs-Homerl-IP3R interaction. Lambert-Eaton myasthenic syndrome (LEMS) is caused by reduced ACh quantal release that occurs mainly because of presynaptic P/Q-type voltage-gated Ca2+ channel (VGCC) antibodies. Physicians should be vigilant for LEMS because it may predict an underlying malignancy, particularly small-cell lung carcinoma; SOX-1 antibodies are usually present in these patients and are absent in those who do not have caucer. Some patients with LEMS have antibodies against synaptotagmin-1, which associates with SNARE complex and functions as Ca2+ sensor for exocytosis. The stimulation of the M1-type presynaptic muscarinic AChR (mAChR)(G-proterin-coupled receptor) can compensate for the deficiency of Ca2+-mediated ACh quantal release via the PLC/DAG-mediated mechanism; This acts in a manner similar to the BDNF/NT4-TrkB interaction. The detection of M1 mAChR antibodies in LEMS suggests an impaired compensatory mechanism and corresponds, at least in part, to autonomic symptoms. Congenital myasthenic syndromes are classified into presynaptic, synaptic basal lamina and postsynaptic defects.     

Congenital myasthenic syndrome with novel pathogenic variants in the COLQ gene associated with the presence of antibodies to acetylcholine receptors

Congenital myasthenic syndrome (CMS) is a heterogeneous group of inherited disorder which does not associate with anti-acetylcholine receptor (AChR) antibody. The presence of AChR autoantibody is pathogenic and highly sensitive and specific for autoimmune myasthenia gravis (MG). We describe 2 children from unrelated families who presented with hypotonia, ptosis and fatigability in early infancy with anti-AChR antibodies detected via ELISA on 2 separate occasions in the sera. Both were treated as refractory autoimmune MG due to poor clinical response to acetylcholinesterase inhibitor and immunotherapy. In view of the atypical clinical features, genetic studies of CMS were performed and both were confirmed to have novel pathogenic mutations in the COLQ gene. To the best of our knowledge, the presence of anti-AChR antibody in COLQ-related CMS has never been reported in the literature. The clinical presentation of early onset phenotype, and refractoriness to acetylcholinesterase inhibitor and immunotherapy should prompt CMS as a differential diagnosis.     

Pathogenic mechanisms of myasthenia gravis induced by antibodies against muscle-specific kinase]

Antibodies to acetylcholine receptor (AChR) are major cause of the human autoimmune disease, myasthenia gravis (MG). Additionally, autoantibodies against Muscle-specific kinase (MuSK) were found in a proportion of patients with generalized MG. After the identification of MuSK antibodies in MG patients, laboratory test for measuring antibodies to MuSK is now required to confirm the diagnosis of MG and the clinical treatment as well as AChR antibodies. MuSK is critical to the clustering of AChR and plays multiple roles at neuromuscular junctions (NMJ). However, it has been dispute concerning the pathogenicity of MuSK antibodies in muscle weakness of MG, as the experimental autoimmune MG caused by MuSK antibodies was absent. Here we describe the recent progress to understand the pathogenic roles of MuSK antibodies in muscle weakness of experimental animals induced by MuSK protein.     

Antibody dependent cell-mediated cytotoxicity--an additional mechanism in human autoimmune myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease characterised by serum anti-acetylcholine receptor (AChR) antibodies and several pathogenic mechanisms for the action of these antibodies have been elucidated. In this study, we have analysed a possible additional mechanism for these antibodies, namely antibody-dependent cell-mediated cytotoxicity (ADCC). Using as target cells a cell line expressing AChR, we could show an increased ADCC mediated by sera from MG patients. Sera with AChR antibodies induced a higher cytotoxicity than sera from patients without these antibodies or healthy individuals. Sera from MG patients with thymoma induced a higher cytotoxic effect than sera from other patients. There was a strong positive correlation between the concentration of AChR antibodies and cytotoxicity mediated by purified IgG fractions from thymoma patients. In addition, there was a higher cytotoxicity mediated by sera from thymoma patients with extended dinucleotide repeats, (AT)n repeats, in the CTLA-4 gene. ADCC mediated by AChR antibodies may thus be another possible pathogenic mechanism that could operate in MG patients, especially in patients with thymoma.     

Putative role of antireticulin antibody in antiacetylcholine-receptor-antibody-negative myasthenia gravis

It has recently been demonstrated that pathogenic immunoglobulins circulate in the blood of patients with acetylcholine-receptor-antibody (A-AChR)-negative myasthenia gravis (MG). Evidence has been presented that in this form of MG the neuromuscular transmission is impaired by antibodies that bind to endplate determinants other than the AChR. We describe three patients with clinical manifestations of A-AChR-negative MG in whom antibody directed to reticulin (A-Ret) was detected. Antibody directed to reticulin is usually associated with celiac disease; however, none of the patients had symptoms or signs of celiac disease. To our knowledge, the association of A-Ret with A-AChR-negative MG has not been reported before. We postulate that A-Ret might help to differentiate between A-AChR-negative MG and congenital myasthenia. Further studies are needed to determine whether A-Ret plays a pathogenic role in A-AChR-negative MG or should instead be considered as an epiphenomenon.     

Histochemical findings of and fine structural changes in motor endplates in diseases with neuromuscular transmission abnormalities

We herein review the histochemical findings and fine structural changes of motor endplates associated with diseases causing neuromuscular transmission abnormalities. In anti-acetylcholine receptor (AChR) antibody-positive myasthenia gravis (MG), type 2 fiber atrophy is observed, and the motor endplates show a reduction in the nerve terminal area, simplification of the postsynaptic membrane, decreased number of acetylcholine receptors, and deposition of immune complexes. In anti-MuSK antibody-positive MG, the fine structure shows a decrease in the postsynaptic membrane length, but the secondary synaptic cleft is preserved. There is no decrease in the number of AChRs, and there are no deposits of immune complexes at the motor endplates. Patients with Lambert-Eaton myasthenic syndrome show type 2 fiber atrophy, their motor endplates show a decrease in both the mean postsynaptic area and postsynaptic membrane length in the brachial biceps muscle. Congenital myasthenic syndrome with episodic apnea is characterized only by small-sized synaptic vesicles; the postsynaptic area is preserved. In subjects with congenital myasthenic syndrome with acetylcholinesterase deficiency, quantitative electron microscopy reveals a significant decrease in the nerve terminal size and presynaptic membrane length; further, the Schwann cell processes extend into the primary synaptic cleft, and partially or completely occlude the presynaptic membrane. The postsynaptic folds are degenerated, and associated with pinocytotic vesicles and labyrinthine membranous networks. Patients with slow-channel congenital myasthenia syndrome show type 1 fiber predominance, and their junctional folds are typically degenerated with widened synaptic space and loss of AChRs. Patients with AChR deficiency syndrome caused by recessive mutations in AChR subunits also show type 1 fiber predominance, and while most junctional folds are normal, some are simplified and have smaller than normal endplates. Rapsin and MuSK mutations cause type 1 fiber predominance, and the small postsynaptic area is associated with AChR decrease.     

Acetylcholinesterase inhibitors in MG: To be or not to be?

Myasthenia gravis (MG) is an autoimmune disorder usually caused by antibodies against either the acetylcholine receptor (AChR) or muscle‐specific tyrosine kinase (MuSK) at the neuromuscular junction. Neuromuscular transmission failure results in muscle fatigue and weakness that can be treated symptomatically with acetylcholinesterase inhibitors (AChEIs). Long‐term treatment with nonselective AChEIs may have considerable drawbacks; thus, this medication is ideally tapered when strength improves. Patients with AChR antibodies respond beneficially to treatment, whereas patients with MuSK antibodies generally do not. Recently, the selective AChEI EN101, which specifically targets the isoform of “read‐through” AChE (AChE‐R), has been developed and may be of importance for symptomatic relief in AChR‐antibody seropositive MG. This article is a review of the mechanisms, therapeutic effects, and drawbacks, with both old and new AChEIs in MG. Muscle Nerve, 2009     

Myasthenia in SCID mice grafted with myasthenic patient lymphocytes: role of CD4+ and CD8+ cells

OBJECTIVES: Acetylcholine receptor (AChR)-specific CD4+ cells are present in MG patients, and synthesis of the high-affinity immunoglobulin G (IgG) autoantibodies (autoAb) against the muscle AChR that causes MG symptoms requires intervention of CD4+ cells. The role of CD4+ cells in MG pathogenesis has been postulated but never proven. MG patients do not have anti-AChR cytotoxic phenomena, and it has been assumed that CD8+ cells do not have a pathogenic role in MG. However, CD8+ cells may facilitate rodent experimental MG, raising the possibility that CD8+ cells might be necessary also in MG. In this study we examined whether CD4+ and CD8+ cells play a role in the pathogenesis of MG and whether CD4+ cells specific for AChR epitope sequences recognized by most MG patients ("universal" epitopes) drive the synthesis of pathogenic antibodies. METHODS: First we characterized a chimeric human-mouse model of MG in severe combined immunodeficiency (SCID) mice engrafted with blood lymphocytes (BL) from MG patients. We used that model to determine whether CD4+ and CD8+ cells are necessary for transfer of MG symptoms. We engrafted SCID mice intraperitoneum with BL from 19 MG patients and 5 healthy controls. We engrafted some mice with either BL, BL depleted in CD4+ or CD8+ cells from the same patient, or CD4+ depleted BL reconstituted with CD4+ T cells from the same patient, specific for "universal" AChR epitopes or for two unrelated antigens, tetanus and diphtheria toxoids. We tested the mice for myasthenic symptoms for 7 to 18 weeks. RESULTS: Mice transplanted with BL, or CD8+ depleted BL, or CD4+-depleted BL reconstituted with anti-AChR CD4+ cells from MG patients frequently developed myasthenic weakness. The mice had human anti-AChR Ab in the serum and bound to muscle AChR. Mice transplanted with BL from controls, or CD4+-depleted BL from MG patients, or CD4+-depleted BL from an MG patient reconstituted with CD4+ cells specific for tetanus or diphtheria toxoids did not develop myasthenic weakness or anti-AChR Ab. CONCLUSIONS: CD4+ cells are necessary for MG pathogenesis; CD8+ cells may not be. CD4+ cells specific for "universal" AChR epitopes help the synthesis of pathogenic Ab.     

Seronegative generalised myasthenia gravis: clinical features,antibodies, and their targets

Myasthenia gravis (MG) is a well-recognised disorder of neuromuscular transmission that can be diagnosed by the presence of antibodies to the acetylcholine receptor (AChR). However, some patients (about 15%) with generalised MG do not have detectable AChR antibodies. There is some evidence, however, that this "seronegative" MG is an antibody-mediated disorder. Plasma from patients with the disorder seems to contain various distinct humoral factors: IgG antibodies that reversibly inhibit AChR function; a non-IgG (possibly IgM) factor that indirectly inhibits AChR function; and an IgG antibody against the muscle-specific kinase (MuSK). The presence of antibodies against MuSK appears to define a subgroup of patients with seronegative MG who have predominantly localised, in many cases bulbar, muscle weaknesses (face, tongue, pharynx, etc) and reduced response to conventional immunosuppressive treatments. Moreover, muscle wasting may be present, which prevents complete response to these therapies.     

AChR phosphorylation and indirect inhibition of AChR function in seronegative MG

BACKGROUND: Approximately 10% to 20% of patients with autoimmune MG do not have antibodies to the acetylcholine receptor (AChR), so-called seronegative MG (SNMG). IgG antibodies from up to 70% of SNMG patients bind to the muscle-specific receptor tyrosine kinase, MuSK. The plasmas and non-IgG fractions from SNMG patients (and some with AChR antibodies) also contain a factor, perhaps an IgM antibody, that inhibits AChR function, but it is not clear how this factor acts and whether it is related to the MuSK IgG antibodies. METHODS: The authors studied 12 unselected SNMG plasmas and their non-IgG fractions; seven were positive for MuSK IgG antibodies. Ion flux assays, electrophysiology, phosphorylation, and kinase assays were used to look at mechanisms of action. RESULTS: Eight of the 12 plasmas and their non-IgG fractions inhibited AChR function, but the inhibitory activity was transient and did not correlate with the presence of MuSK IgG antibodies. Two of three plasmas added outside of a cell-attached patch pipette inhibited AChR function within the patch, and these two plasmas also increased AChR phosphorylation. CONCLUSIONS: The authors propose that a plasma factor(s) in SNMG patients, distinct from MuSK IgG antibodies, binds to a muscle membrane receptor and activates a second messenger pathway leading to AChR phosphorylation and reduced AChR function. Identifying the target for this factor should lead to improved diagnosis of MG in MuSK antibody-negative patients and may provide new insights into the function of the neuromuscular junction and pathophysiological mechanisms in MG.     

Overlap myasthenic syndrome: combined myasthenia gravis and Eaton-Lambert syndrome

A patient with a known history of pernicious anemia had the combined features of autoimmune myasthenia gravis (MG) and the Eaton-Lambert syndrome (ELS). Initially, this patient had all the features typical of MG, and after thymectomy developed all the typical features of ELS. In view of the coexistence of two autoimmune neuromuscular transmission disorders in one patient, we termed this disorder "overlap myasthenic syndrome."     

Neurophysiologic assessment of disorders affecting the neuromuscular junction

The neuromuscular junction (NMJ) is a specialized synapse with a complex structural organization. Muscle contraction involves several steps: (1) nerve conduction to depolarize the motor nerve terminals, (2) opening of voltage-gated calcium channels (VGCCs) in the presynaptic membrane, (3) generation of endplate potential in the postsynaptic membrane via acetylcholine receptors, (4) depolarization of muscle sodium channels, and (5) excitation-contraction (E-C) coupling. Each step can be affected by various diseases. Guillain-Barre syndrome involves distal axons and possibly the presynaptic NMJ. The abnormalities can be detected by nerve conduction studies and single-fiber electromyography (SFEMG). Myasthenia gravis (MG) with anti-acetylcholine receptor (AChR) antibodies, is the most common NMJ disorder, and ~5% of myasthenia patients are positive for anti-muscle specific kinase (MuSK) antibodies. Patterns and severity of neuromuscular transmission failure detected by repetitive nerve stimulation test and SFEMG are somewhat different in AChR-MG and MuSK-MG. Excitation-contraction (E-C) coupling can be affected by MG, possibly via antibodies against ryanodine receptors. The E-C coupling time can be assessed with an accelerometer. Lambert-Eaton myasthenic syndrome is caused by antibodies against presynaptic VGCCs. This review will focus on neurophysiological testing, including SFEMG, and measurements of E-C coupling time with an accelerometer. In addition to confirming or excluding the diagnosis, these techniques can provide new insights into the pathophysiology of a variety of neuromuscular disorders.